Culturing of primary hepatocytes as entrapped aggregates in a packed bed bioreactor: A potential bioartificial liver

Summary Conventional culture systems for hepatocytes generally involve cells cultured as flat, monolayer cells, with limited cell-cell contact, in a static pool of medium, unlike the liver in vivo where the parenchymal cells are cuboidal, with extensive cell-cell contact, and are continuously perfused with blood. We report here a novel bioreactor system for the culturing of primary hepatocytes with cuboidal cell shape, extensive cell-cell contact, and perfusing medium. The hepatocytes were inoculated into the bioreactor and allowed to recirculate at a rate optimal for them to collide and form aggregates. These newly-formed aggregates were subsequently entrapped in a packed bed of glass beads. The bioreactor was perfused with oxygenated nutrient medium, with controlled oxygen tension, pH, and medium perfusion rate. The hepatocytes were viable for up to the longest time point studied of 15 days in culture based on urea synthesis, albumin synthesis and cell morphology. Light microscopy studies of hepatocytes cultured for 15 days in the bioreactor showed interconnecting three-dimensional structures resembling the hepatic cell plate in the liver organ. Electron microscopy studies on the same cells revealed ultrastructure similar to the hepatocytes in vivo, including the presence of plentiful mitochondria, rough and smooth endoplasmic reticulum, glycogen granules, peroxisomes, and desmosomes. We believe that our hepatocyte bioreactor is a major improvement over conventional culture systems, with important industrial applications including toxicology, drug metabolism, and protein/peptide synthesis. The hepatocyte bioreactor concept may also be used as the basis for the development of a bioartificial liver to provide extracorporeal hepatic support to patients with hepatic failure.     

Stimulation of DNA synthesis and mitotic activity of chick embryo hepatocytes in primary culture

Summary Monolayer cultures were prepared from hepatocytes of 15 d chick embryos and maintained at high cell density in a chemically defined medium. In the absence of growth stimulatory conditions DNA synthesis was observed only during the first 10 to 16 h of culture. Thus, after a 12 h exposure to [³H]thymidine ([³H]dThd, 4 to 16 h) 9.1±1% ( ,n=4) of the hepatocyte nuclei were labeled. Labeled mitotic nuclei, up to late telophase, were regularly observed in these cultures. Beyond 16 h less than 2% labeled nuclei were found (12 h of [³H]dThd), which indicates that the hepatocytes entered proliferative quiescence. DNA synthesis of “resting” hepatocytes was stimulated by insulin and, only slightly, by hydrocortisone, glucagon, or fetal bovine serum. Triiodothyronine (T₃), or the nucleoside inosine (i) did not stimulate. Combination of insulin (I) with hydrocortisone (H), T₃ (T), or glucagon (G) resulted in a more than additive effect. Nearly maximal stimulation occurred with the combinations IHT and ITG. Labeling increased at 10 ng/ml of each component and was maximal at 1 to 10 μg/ml. A lag period of 8 to 10 h after hormone administration (IHiTG, 10 μg/ml) was observed before nuclear labeling increased. Within the subsequent 10 h a considerable proportion of the hepatocytes (up to 30% or more) entered DNA synthesis. Mitotic activity (with nuclei in prophase up to late telophase) also was stimulated. An increase of both total DNA and protein content was measured in several experiments. Hormonal stimulation of hepatocyte DNA synthesis and mitotic activity was associated with decreased β-naphthoflavone-mediated induction of cytochrome P450. A causal relationship between these two phenomena remains to be established. It is suggested that chick embryo hepatocyte cultures are a useful tool for studies on hepatocyte proliferation and differentiation. The present study is based on original observations by Dr. F. R. Althaus (presently at the Institute of Pharmacology and Biochemistry, University of Zürich, Switzerland). This contribution of his and his incisive criticism are acknowledged. The study was supported by Grant 3.893.81 from the Swiss National Research Foundation.     

Epidermal growth factor receptor numbers in male and female mouse primary hepatocyte cultures

Epidermal growth factor receptors (EGF-R) were measured in adult male and female mouse primary hepatocyte cultures. On culture day 1, female hepatocytes had significantly fewer EGF-R than male hepatocytes (1.3 x 10(4) versus 6.2 x 10(5) per cell). Over the next three days, morphological changes consistent with progressive heptocyte dedifferentiation were observed. During this period, EGF-R numbers progressively increased in female cultures and decreased in male cultures, and by day 4 the sexual difference in EGF-R numbers was obliterated. These results indicate that a relationship exists between the degree of differentiation in hepatocyte cultures and the expression of EGF-R on the cell surface.     

Fractionation of Rat Hepatocyte Subpopulations with Varying Metabolic Potential, Proliferative Capacity, and Retroviral Gene Transfer Efficiency

The liver contains hepatocytes with varying ploidy and gene expression. To isolate cells on the basis of ploidy for analyzing mechanisms concerning cell proliferation and differentiation, we used Percoll gradients to separate F344 rat hepatocyte subpopulations. Specific fractions were enriched in polyploid (H2 fraction) or diploid (H3 and H4 fractions) hepatocytes containing glycogen and glucose-6-phosphatase. H4 cells were relatively smaller with greater nuclear/cytoplasmic ratios, less complex cytoplasm, and higher serum albumin or ceruloplasmin biosynthetic rates. H2 fraction cells were larger with lesser nuclear/cytoplasmic ratio, more complex cytoplasm, and more cytochrome P450 activity. Phenotypic marking showed that H4 cells originated in zone one and H2 cells in zones two or three of the liver lobule. H4 cells showed much greater mitogenic responsiveness to human hepatocyte growth factor. Retroviral gene transfer, which requires both viral receptors and cellular DNA synthesis, was significantly more efficient in H4 cells. The findings indicated thatsmalldiploid andlargepolyploid hepatocytes show unique biological differences. The ability to isolate hepatocytes of varying maturity is relevant for mechanisms concerning liver growth control and hepatic gene expression.     

肝细胞生长因子对四氯化碳损伤原代培养大鼠肝细胞的保护作用

本工作采用无血清原代培养大鼠肝细胞法,观察了重组人肝细胞生长因子（ｒ－ｈＨＧＦ）对四氯化碳（ＣＣｌ４）致大鼠肝细胞损伤的保护作用。结果表明,ｒ－ｈＨＧＦ对ＣＣｌ４染毒肝细胞有明显的保护作用。ｒ－ｈＨＧＦ保护组较ＣＣｌ４染毒组细胞存活率显著升高,细胞内丙氨酸转氨酶、钾离子漏出明显降低。结果提示,ｒ－ｈＨＧＦ可减轻ＣＣｌ４对肝细胞膜的损伤,提高细胞膜的结构完整性     

Engineering hepatocyte functional fate through growth factor dynamics: the role of cell morphologic priming.

We have reported previously that cellular stimulation induced by variable mechanochemical properties of the extracellular microenvironment can significantly alter liver-specific function in cultured hepatocytes (Semler et al., Biotech Bioeng 69:359-369, 2000). Cell activation via time-invariant presentation of biochemical growth factors was found to either enhance or repress cellular differentiation of cultured hepatocytes depending on the mechanical properties of the underlying substrate. In this work, we investigated the effects of dynamic growth factor stimulation on the cell growth and differentiation behavior of hepatocytes cultured on either compliant or rigid substrates. Specifically, hepatotrophic growth factors (epidermal and hepatocyte) were either temporally added or withdrawn from hepatocyte cultures on Matrigel that was crosslinked to yield differential degrees of mechanical compliance. We determined that the functional responsiveness of hepatocytes to fluctuations in GF stimulation is substrate specific but only in conditions in which the initial mechanochemical environment induced significant cell morphogenesis. Our studies indicate that in conditions under which hepatocytes adopted a "rounded" phenotype, they exhibited increased levels of differentiated function upon soluble stimulation and markedly decreased function upon the depletion of GF stimulation. In contrast, hepatocytes that assumed a "spread" phenotype exhibited slightly increased function upon the depletion of GF stimulation. By examining the functional responsiveness of hepatocytes of differential morphology to varied fluctuations in GF activation, insights into the ability of cell shape to "prime" hepatocyte behavior in dynamic microenvironments were elucidated. We report on the possibility of uncoupling and, thus, selectively manipulating, the concerted contributions of GF-induced cellular activation and substrate- and GF-induced cell morphogenesis toward induction of cell function.     

Colony isolation and secondary culture of fetal porcine hepatocytes on STO feeder cells

Summary The secondary culture of non-transformed parenchymal hepatocytes has not been possible. STO feeder cell-dependent secondary cultures of fetal pig hepatocytes were established by colony isolation from primary cultures of 26-d fetal livers. The liver cells had the typical polygonal morphology of parenchymal hepatocytes. They also spontaneously differentiated to form small biliary canaliculi between individual cells or progressed further to large multicellular duct-like structures or cells undergoing gross lipid accumulation and secretion. The secondary hepatocyte cultures expressed alpha-fetoprotein (AFP), albumin, and β-fibrinogen mRNA, and conditioned medium from the cells contained elevated levels of transferrin and albumin. STO feeder cell co-culture may be useful for the sustainable culture of hepatocytes from other species.     

Immortalization of chimpanzee hepatocytes with an amphoteric retrovirus encoding simian virus 40 T antigen.

Primary chimpanzee (Pan troglodytes) hepatocyte cultures were maintained in a serum-free medium containing hormones and growth factors and exhibited the de novo synthesis and secretion of numerous liver-specific plasma proteins for over 3 weeks in vitro. The long-term maintenance of differentiated, primate hepatocytes in this serum-free medium allowed for subsequent immortalization events to occur after infection with the amphoteric retrovirus U19, which encodes the simian virus 40 large T antigen oncogene. Several hepatocyte cell lines were selected and examined for the expression of liver-specific plasma proteins and the capacity to synthesize apolipoproteins. Several cell lines expressed a majority of the plasma proteins investigated, including apolipoproteins A1 and E. These results demonstrate the ability of this serum-free medium to maintain long-term differentiated primate hepatocytes, allowing for the experimental immortalization of this cell type in vitro and the maintenance of differentiated functions in the established cell lines. This methodology should be amenable to the study of the liver and its related diseases.     

Neutral red (NR) assay for cell viability and xenobiotic-induced cytotoxicity in primary cultures of human and rat hepatocytes

Neutral red (NR) in medium was absorbed and concentrated in lysosomes of cultured rat and human hepatocytes. NR uptake increased with the time of incubation and reached a plateau in 2 hr. Uptake was proportional to the concentration of the NR solution and the numbers of viable liver cells. Prolonged culture of hepatocytes increased the numbers of lysosomes, and thus, the dye accumulation. The NR can be extracted from lysosomes for quantitative measurement of hepatocyte viability and cytotoxicity of xenobiotics. With this assay, several serum-free media (e.g., Waymouth's, MEM, LHC-8, etc.) were compared for the maintenance of viable hepatocytes in vitro. Interestingly, LHC-8 medium, which is used to grow human bronchial epithelial cells, best preserved viable rat hepatocytes. The cytotoxic effects of dimethylnitrosamine (DMN) and aflatoxin B₁ (AFB₁) were examined by NR assay on rat and human hepatocyte cultures and were found to be dependent on dose and time of the exposures. NR₅₀ was 20 mM for DMN and 0.072 µM for AFB₁ in rat hepatocytes with 24 hr of exposures and reduced to 12.5 mM for DMN and 0.053 µ uM for AFB₁ with 48 fr exposures. Human hepatocytes were more resistant to the toxicity of both chemicals; NR₅₀ values were 100 mM DMN and 1.8 µM AFB₁ respectively, for 24 hr treatments. Compared with lactate dehydrogenase (LDH) leakage test, the NR assay was simpler and more sensitive in determining the viability and cytotoxicity of xenobiotics in primary cultures of hepatocytes.Abbreviations NR Neutral Red - MEM Eagle's Minimum Essential Medium - DMN dimethylnitrosamine - AFB₁ aflatoxin B₁ - LDH lactate dehydrogenase - HBSS Hanks balanced salt solution; - EDTA ethylene bis (oxyethylenenitrilo)-tetraacetic acid - L-15 Leibovitz's 15 - NADH B-nicotinamide adenine dinu - FBS fetal bovine serum - IA immediate autopsy Contribution No. 2816 from Laboratory of Genotoxicology.     

Intermittent addition of HGF and TGF-β1 in rat primary hepatocyte culture

The effects of hepatocyte growth factor (HGF) and transforming growth factor-beta (TGF-β) on two morphological states of hepatocytes in monolayer and spheroid cultures, were examined in terms of their mitogenic ability and albumin expression. In monolayer culture on collagen-coated dishes, the increase in DNA content in the presence of HGF was observed when HGF was added within two days of cell isolation, whereas no increase in DNA was observed when HGF was added four days of cell isolation. DNA content increased even after four days, when HGF was added intermittently. On the other hand, spheroid formation was promoted on Primaria® dishes in HGF-free culture, whereas it was inhibited following the addition of HGF. No increase in DNA content was observed in spheroid cultures even in the presence of HGF throughout the culture period. The albumin production ability rapidly decreased in monolayer culture, but the decline was attenuated following the addition of HGF during the course of culture. A high albumin production ability was maintained independent of HGF supplementation in spheroid culture. Both DNA content and albumin production decreased rapidly following the addition of TGF-β1 in monolayer culture, and this decline was also attenuated following the addition of HGF to the medium.     

Evaluation and optimization of hepatocyte culture media factors by design of experiments (DoE) methodology

Optimization of cell culture media based on statistical experimental design methodology is a widely used approach for improving cultivation conditions. We applied this methodology to refine the composition of an established culture medium for growth of a human hepatoma cell line, C3A. A selection of growth factors and nutrient supplements were systematically screened according to standard design of experiments (DoE) procedures. The results of the screening indicated that the medium additives hepatocyte growth factor, oncostatin M, and fibroblast growth factor 4 significantly influenced the metabolic activities of the C3A cell line. Surface response methodology revealed that the optimum levels for these factors were 30 ng/ml for hepatocyte growth factor and 35 ng/ml for oncostatin M. Additional experiments on primary human hepatocyte cultures showed high variance in metabolic activities between cells from different individuals, making determination of optimal levels of factors more difficult. Still, it was possible to conclude that hepatocyte growth factor, epidermal growth factor, and oncostatin M had decisive effects on the metabolic functions of primary human hepatocytes.     

The importance of physiological oxygen concentrations in the sandwich cultures of rat hepatocytes on gas‐permeable membranes

Oxygen supply is a critical issue in the optimization of in vitro hepatocyte microenvironments. Although several strategies have been developed to balance complex oxygen requirements, these techniques are not able to accurately meet the cellular oxygen demand. Indeed, neither the actual oxygen concentration encountered by cells nor the cellular oxygen consumption rates (OCR) was assessed. The aim of this study is to define appropriate oxygen conditions at the cell level that could accurately match the OCR and allow hepatocytes to maintain liver specific functions in a normoxic environment. Matrigel overlaid rat hepatocytes were cultured on the polydimethylsiloxane (PDMS) membranes under either atmospheric oxygen concentration [20%‐O₂ (+)] or physiological oxygen concentrations [10%‐O₂ (+), 5%‐O₂ (+)], respectively, to investigate the effects of various oxygen concentrations on the efficient functioning of hepatocytes. In parallel, the gas‐impermeable cultures (polystyrene) with PDMS membrane inserts were used as the control groups [PS‐O₂ (?)]. The results indicated that the hepatocytes under 10%‐O₂ (+) exhibited improved survival and maintenance of metabolic activities and functional polarization. The dramatic elevation of cellular OCR up to the in vivo liver rate proposed a normoxic environment for hepatocytes, especially when comparing with PS‐O₂ (?) cultures, in which the cells generally tolerated hypoxia. Additionally, the expression levels of 84 drug‐metabolism genes were the closest to physiological levels. In conclusion, this study clearly shows the benefit of long‐term culture of hepatocytes at physiological oxygen concentration, and indicates on an oxygen‐permeable membrane system to provide a simple method for in vitro studies. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1401–1410, 2014     

Hepatocyte-specific porous polymer-scaffolds of alginate/galactosylated chitosan sponge for liver-tissue engineering

Porous scaffolds of alginate/galactosylated chitosan (ALG/GC) sponges were prepared by lyophilization for liver-tissue engineering. Primary hepatocytes in ALG/GC sponges showed higher cell attachment and viability than in alginate alone owing to the specific interaction of the asialoglycoprotein receptors on hepatocyte with the galactose residues on ALG/GC sponges. Improvements in spheroid formation and long-term liver-specific functions of the immobilized hepatocyte were also observed in ALG/GC sponge.     

Multilayer rat hepatocyte aggregates formed on expanded polytetrafluoroethylene surface

Feasibility of using a macroporous membrane material, expanded polytetrafluoroethylene (ePTFE), for culturing hepatocytes on its surface was examined. Adult rat hepatocytes were attached to an ePTFE surface and cultured in a hormonally defined medium supplemented with or without fetal calf serum (FCS, 10%) or bovine serum albumin (BSA, 0.03–3%). When cultured in a FCS-suplemented medium, hepatocytes reorganized themselves into multilayer cell aggregates on an ePTFE surface. The morphological characteristics of hepatocytes were influenced by the modification of the ePTFE surface as well as the culture medium. Hepatocytes cultured on a polyvinylalcohol (PVA)-coated ePTFE surface formed many more multilayer cell aggregates than those cultured on an uncoated ePTFE surface. Such highly multilayered hepatocyte aggregates were also noted when the cells were cultivated in a BSA-supplemented medium. On the other hand, when cultured in a FCS- or BSA-free medium, hepatocytes formed cell monolayers on both PVA-coated and uncoated ePTFE surfaces as did the cells on a collagen-coated polystyrene surface. The hepatocytes in the aggregates exhibited high albumin expression capability and low DNA synthesis rate as compared with those in monolayer cultures. The multilayer hepatocyte aggregates, as immobilized on a PVA-coated ePTFE surface in a serum-supplemented medium, are shown to be not only morphologically, but functionally differentiated, and will provide us a model system for the development of a bioreactor using hepatocytes, particularly for a hybrid-type artificial liver. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterization of the effects of human placental HGF on rat hepatocytes.

Hepatocyte growth factor (HGF) also known as hepatopoietin A (HPTA) (Michalopoulos, FASEB J., 4:176-187, 1990) is a heparin-binding growth factor whose characterization and tissue distribution have been reported elsewhere. This growth factor was recently cloned and its amino acid sequence determined under the name of hepatocyte growth factor (HGF) (Miyazawa et al., Biochem. Biophys. Res. Commun., 163:967-973, 1989; Zarnegar et al., Biochem. Biophys. Res. Commun., 163:1370-1376, 1989; Nakamura et al., Nature, 342:440-443, 1989). Human placenta is one of the tissues that contains significant amounts of HGF. We isolated HGF from human placenta and characterized its biologic effect on rat hepatocytes. Human placenta HGF was isolated in high purity as a single chain molecule. Single chain HGF stimulated DNA synthesis in primary rat hepatocyte cultures in serum-free medium. The maximal effect was seen at 5-10 ng/ml. The maximal response occurred at 25-48 hours after plating of the hepatocytes. Protein synthesis was also stimulated by HGF in primary rat hepatocyte cultures. There were peak responses at 19-24 and 37-42 hours after plating of the hepatocytes. TGF beta 1 inhibited more than 95% of HGF-induced DNA synthesis but only 25% of HGF-induced protein synthesis. HGF interacted in an additive manner with EGF, a well-known hepatocyte mitogen. There was not an additive interaction between HGF and aFGF. Regenerating liver hepatocytes obtained from rats which underwent two-thirds partial hepatectomies (PHX) also responded to HGF in a dose-dependent manner as the hepatocytes from normal liver.     

The bicarbonate ion is essential for efficient DNA synthesis by primary cultured rat hepatocytes

Summary Bicarbonate in the culture medium is essential for DNA synthesis of primary cultured rat hepatocytes stimulated by epidermal growth factor (EGF). When primary cultured hepatocytes in supplemented Leibovitz L15 medium were placed in a 100% air incubator, no increase in DNA synthesis was observed even after stimulation by EGF. However, when these cells were cultured with NaHCO₃ and EGF and placed in a 5% CO₂:95% air incubator, a stimulus of DNA synthesis more than 10-fold greater than in cultures in air only was seen, and many mitotic figures could be identified. Furthermore, NaHCO₃ added to supplemented DMEM/F12 medium enhanced the DNA synthesis of primary cultured rat hepatocytes in this medium. The ideal pH of the medium for DNA synthesis of cultured hepatocytes was in the range of 7.6 to 8.0. A dose response of NaHCO₃ in several media showed that DNA synthesis of the cells increased as the concentration of NaHCO₃ increased and that 25 to 30 mM NaHCO₃ in the medium was optimal for the replication of DNA by primary cultured rat hepatocytes. The investigations described in this study were supported in part by grants CA-07175, CA-22484, and CA-45700 from the National Cancer Institute, Bethesda, MD.     

Formation of multilayer aggregates of hepatocytes cultured on ligand-coated surfaces

Hepatocytes grown on various ligand (hepatocyte growth factor, HGF; low-density lipoprotein, LDL; transferrin; poly-N-p-vinylbenzyl-d-lactonamide, PVLA)-coated surfaces tended to keep round cell shapes and form multilayer aggregates in the presence of hepatocyte growth factor. Inhibitors of proliferating signal transduction did not affect mitogen-induced hepatocyte aggregation, whereas colchicine, an antimicrotubular drug, completely inhibited the hepatocyte aggregation. Meanwhile, significant ligand loss was found on hepatocytes moving over surfaces and thus receptor-mediated endocytosis might be directly involved in the hepatocyte aggregation on ligand-coated surfaces.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin inhibits DNA synthesis in rat primary hepatocytes

Treatment of Sprague-Dawley (SD) rats with a dosing regimen of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) maintaining a steady-state liver concentration of 150 ng/g results in enhanced hepatocyte proliferation in the periportal region, but reduced proliferation in the remainder of the hepatic lobule (Fox et al. (1993) Cancer Res., 53, 2265–2271). Here, we report an initial characterization of the actions of TCDD on hepatocyte proliferation by monitoring DNA synthesis in primary hepatocytes isolated from SD rats. TCDD caused a dose-dependent inhibition (EC₅₀ = 10 pM) of DNA synthesis in primary hepatocytes isolated from either male or female SD rats in the presence or absence of known hepatocyte mitogens (epidermal growth factor, hepatocyte growth factor, and transforming growth factor ). No change in DNA synthesis was observed at TCDD concentrations less than 1 pM. Initial characterization of the EGF response system in these cells revealed that TCDD did not alter the specific binding of EGF, or the levels of EGF receptor protein measured in intact cells or cell lysates. TCDD-dependent inhibition of DNA synthesis occurred independently of the suppression observed with transforming growth factor-β1. Estradiol did not alter DNA synthesis in the presence or absence of TCDD. Taken together, these findings indicate that TCDD suppresses DNA synthesis via a novel pathway that is non-responsive to estradiol, independent of TGF-β, and does not involve a decreased ability of hepatocytes to recognize (bind) EGF, a prototype mitogen.     

Formation of bile canaliculi in long-term primary cultures of adult rat hepatocytes on permeable membrane: an ultrastructural study

Adult rat hepatocytes were cultured for 15 days on type I collagen-coated permeable membranes in a hormonally defined Waxman's modified medium supplemented with very low concentrations of insulin, glucagon and dexamethasone. Phase contrast examination showed that 15-day-old cultures still formed a regular monolayer of polygonal cells. In similarly aged cultures, intracellular glycogen was abundant and evenly distributed, while steatosis remained very limited. Scanning and transmission electron microscopy showed that well developed bile canaliculi could be observed on the lateral side of the hepatocyte membrane after 4 days of incubation and persisted for 2 weeks. These canalicular structures probably originated from coalescence of membrane invaginations observed in 1-day-old cultures. Transmission electron microscopy showed that the ultrastructure of the cells was very close to that of normal rat hepatocytes in the intact liver. These results suggest that rat hepatocytes cultured under these experimental conditions are able to develop and maintain tissue-specific cytochemical and morphological properties for at least 15 days.